The relationship between tissue concentrations of adenine nucleotides (ANs) at the end of cold preservation period and organ viability after transplantation is controversial. The purpose of this study is to examine energy metabolism of the pancreas graft preserved by a two-layer cold storage method and the relationship with the viability of the pancreas graft following transplantation. After preservation by simple cold storage with Euro-Collins' solution (EC) (group 1) or University of Wisconsin solution (UW) (group 2), an original two-layer (EC/perfluorochemical(PFC)) method (group 3) and a modified two-layer (UW/PFC) method (group 4) for 24, 48, 72, 96 and 120 hours, tissue concentrations of ANs were determined using high-performance liquid chromatography (HPLC) and the viability of the pancreas graft was tested in the canine model of segmental pancreas autotransplantation. While EC alone (group 1) was available only for 24-hour preservation, UW alone (group 2) and the original two-layer method (group 3) were effective up to 72-hour preservation and the modified two-layer method (group 4) was successful up to 96-hour preservation. In simple cold storage with EC or UW, there was no significant difference between tissue concentrations of adenosine triphosphate (ATP) of viable grafts and nonviable grafts. But in groups 3 and 4, there was an excellent correlation between the posttransplant viability and ATP tissue concentration at the end of preservation, and ATP was useful to predict the viability of the canine pancreas during preservation by the two-layer method before transplantation.